Research Summary Current FDA-approved drugs are usually either small molecules (MW <500) or large proteins (MW >5000). Small molecules are generally limited to targeting proteins and other biomolecules that contain deep binding pockets (e.g., enzymes and GPCRs), which represent ~10% of all disease relevant human proteins. On the other hand, biologics (e.g., monoclonal antibodies) are restricted to extracellular targets, which represent another ~10% of all drug targets. The remaining ~80% drug targets, which are primarily proteins involved in intracellular protein-protein interactions (PPIs), are currently undruggable by either approach. The same limitations apply to the use of small molecules and proteins as research tools. The overall goal of my research is to develop a general approach to targeting the ~80% undruggable proteins. Recent work from my lab as well as many other laboratories has demonstrated that mono- and bicyclic peptides in the 700-2000 molecular-weight range act as effective PPI inhibitors. My group further demonstrated that by integrating a newly discovered class of cyclic cell-penetrating peptides (CPPs) into our compound design, we can generate cell-permeable and metabolically stable cyclic peptide inhibitors against a wide variety of intracellular enzymes and PPIs. During the next five years, we plan to further investigate the mechanism of action of these cyclic CPPs and use the knowledge to develop additional CPPs of improved properties, e.g., CPPs with specificity for tumor tissues. The CPPs will be explored for delivery of proteins as therapeutics and research tools. Efforts will also be made to optimize the potency, selectivity, metabolic stability, and other drug properties of cyclic peptide inhibitors already discovered against several important drug targets including calcineurin, CAL PDZ domain, and NEMO. Finally, we plan to develop a novel methodology for synthesizing and screening large libraries of non-peptidyl macrocycles that can passively diffuse into mammalian cells and act as inhibitors against intracellular proteins such as PPIs.